A Randomised Placebo-Controlled Trial of a Traditional Chinese Herbal Formula in the Treatment of Primary Dysmenorrhoea

Background Most traditional Chinese herbal formulas consist of at least four herbs. Four-Agents-Decoction (Si Wu Tang) is a documented eight hundred year old formula containing four herbs and has been widely used to relieve menstrual discomfort in Taiwan. However, no specific effect had been systematically evaluated. We applied Western methodology to assess its effectiveness and safety for primary dysmenorrhoea and to evaluate the compliance and feasibility for a future trial. Methodology/Principal Findings A randomised, double-blind, placebo-controlled, pilot clinical trial was conducted in an ad hoc clinic setting at a teaching hospital in Taipei, Taiwan. Seventy-eight primary dysmenorrheic young women were enrolled after 326 women with self-reported menstrual discomfort in the Taipei metropolitan area of Taiwan were screened by a questionnaire and subsequently diagnosed by two gynaecologists concurrently with pelvic ultrasonography. A dosage of 15 odorless capsules daily for five days starting from the onset of bleeding or pain was administered. Participants were followed with two to four cycles for an initial washout interval, one to two baseline cycles, three to four treatment cycles, and three follow-up cycles. Study outcome was pain intensity measured by using unmarked horizontal visual analog pain scale in an online daily diary submitted directly by the participants for 5 days starting from the onset of bleeding or pain of each menstrual cycle. Overall-pain was the average pain intensity among days in pain and peak-pain was the maximal single-day pain intensity. At the end of treatment, both the overall-pain and peak-pain decreased in the Four-Agents-Decoction (Si Wu Tang) group and increased in the placebo group; however, the differences between the two groups were not statistically significant. The trends persisted to follow-up phase. Statistically significant differences in both peak-pain and overall-pain appeared in the first follow-up cycle, at which the reduced peak-pain in the Four-Agents-Decoction (Si Wu Tang) group did not differ significantly by treatment length. However, the reduced peak-pain did differ profoundly among women treated for four menstrual cycles (2.69 (2.06) cm, mean (standard deviation), for the 20 women with Four-Agents-Decoction and 4.68 (3.16) for the 22 women with placebo, p = .020.) There was no difference in adverse symptoms between the Four-Agents-Decoction (Si Wu Tang) and placebo groups. Conclusion/significance Four-Agents-Decoction (Si Wu Tang) therapy in this pilot post-market clinical trial, while meeting the standards of conventional medicine, showed no statistically significant difference in reducing menstrual pain intensity of primary dysmenorrhoea at the end of treatment. Its use, with our dosage regimen and treatment length, was not associated with adverse reactions. The finding of statistically significant pain-reducing effect in the first follow-up cycle was unexpected and warrants further study. A larger similar trial among primary dysmenorrheic young women with longer treatment phase and multiple batched study products can determine the definitive efficacy of this historically documented formula. Trial Registration Controlled-Trials.com ISRCTN23374750

Finding effective traditional Chinese herbal medicine has now become one of the interests of medicine, pharmacology, and agricultural biotechnology worldwide. Among the thousands of herbals, some herbs have been freely used among Chinese. Formula or prescriptions listed in the ancient pharmacopoeia of traditional Chinese medicine are usually composed by 3-5 herbs for treatment of illnesses. However, there are no consistent and systematic evidence-based data to support their efficacy or precise biochemical analysis of their complicated compositions. This is partly due to the lack of uniformity of the source of the herbs, the method of their production, and the method of preparation of the concoctions. The gold standard of obtaining evidence for efficacy is to conduct a randomized double-blind placebo-controlled clinical trial. However, the problem arises about the selection of safe herbs or prescriptions to be tested in human experiments. The survey of the prevalence of the use of Chinese medicines among the employees of a research institution in August 2000 found that four-agents-decoction was ranked first among the named Chinese medicine that had been most frequently used in the previous year and the usage was for prophylaxis and relief of menstrual pain. Most of the users obtained the materials without consulting traditional Chinese medicine practitioners. No reports of adverse reactions or warning in use of four-agentsdecoction have ever been published; either did the users report in this survey.
Four-agents-decoction consists of four herbs, prepared Radix Rehmanniae praeparata (熟 地黃), Radix Angelicae Sinensis (當歸), Radix Paeoniae Alba (白芍), and Rhizoma Ligustici Chuanxiong (川芎), which has been used as a basic formula in traditional Chinese medicine since Sung Dynasty. Women's illnesses are among the most commonly treated with traditional Chinese medicine. A few clinical studies conducted in other countries have tested or are testing similar herbs or formulas containing herbs present in four-agents-decoction for the relief of dysmenorrhea. Four-agents-decoction can thus be considered a safe formula; there should not be any delay of conducting a randomized double-blind placebo-controlled parallel intervention trial of four-agents-decoction for dysmenorrhea in Taiwan. This is a pragmatically oriented pilot trial incorporated with an observational epidemiologic study. Subjects are healthy young college women who have suffered from primary dysmenorrhea for at least four consecutive times for the past six cycles will be recruited from Chinese Institute of Technology and Takming College. The research activities include recruiting, randomizing, washing-out, establishing baseline, treating subjects with four-agentsdecoction and placebo for three cycles, and following-up three cycles for the assessment of menstrual pain. Examinations for screening and monthly check up are at the Taipei Municipal Chung Hsiao Hospital for women from Chinese Institute of Technology and at the Tri-Service General Hospital for women from Takming College. At the start and end of the trial, five traditional Chinese medicine practitioners will examine signs and symptoms independently and concurrently on each of the subjects using a standardized checklist. The scores of the symptoms and signs will be further evaluated for the validity of differentiated patterns in determination of the efficacy of four-agents-decoction.
This intervention trial (FADD) will be conducted in collaboration with other nine specialists, including three gynecologists, four traditional Chinese medicine physicians, one pharmacologist, and one statistician. An estimate of 15 months is needed to complete this trial.

五、試驗目的及背景說明(包括學理根據及有關文獻報告)：
Four-agents-decoction, containing prepared Radix Rehmanniae praeparata (熟 地黃), Radix Angelicae Sinensis (當歸), Radix Paeoniae Alba (白芍), and Rhizoma Ligustici Chuanxiong (川芎), is an ancient formula in the treatment of women illnesses since Sung Dynesty. This theory was recently emphasized in Taiwan (1), mainland China (2), and Japan(3). A small scale clinical study conducted in mainland China (4) has shown the effectiveness of four-agents-decoction for prophylaxes or treatment of menstrual disorder. Of the 40 women between age of 14 and 50 who met the criteria of dysmehorrhea, 24 women (60%) after used the prescription had normal menstrual length and amount or the pain stopped. Another 12 women (30%) were improved to some degrees or had less pain. There was no recurrence of pain even three months after the treatment. Although no specific warnings in use of this aged formula were ever reported (5), the efficacy of this formula for treating dysmenorrhea has never been studied in a randomized, double-blind placebocontrolled clinical trial.
Working women in Taiwan can be granted one sick day off each month due to dysmenorrhea. In a sample of college women the prevalence rate of dysmenorrhea was 82% (unpublished data); the pain intensity ranged from slight pain, pain, to severe pain on fourpoint pain scale. The morbidity of dysmenorrhea had been hidden and private, but now it becomes a challenge to public health. While the effectiveness of oral contraceptives or analgesics for relieving menstrual pain is evident in general practice of Western medicine, there is a subset of women who do not respond to this type of conventional treatment and seek althernative medicine or therapy (6) . A variety of basic formulas with substracted and added materials (加減方) and fixed materials in single formulas (固定方) has been available in traditional Chinese medicine (7), in which prepared Radix Rehmanniae Praeparata (熟地 黃), Radix Angelicae Sinensis (當歸), Radix Paeoniae Alba (白芍), and Rhizoma Ligustici Chuanxiong (川芎) are the major materials in 40 of the formulas (8). Starting a week prior to and until the onset of menstruation as a preventive measure has been suggested for an effective relief of the pain (9-10). Over 60% of dysmenoric students in the Chinese Technology Insitute have used this remedy in a variety of forms for prophylaxis to menstrual discomfort (unpublished data). Four-agents-decoction in powder or tablets is reimbursed by the National Health Insurance Bureau. However, the efficacy of this formula has never been evaluated, either has the dosage. Knowledge of the efficacy based on clinical evidence will be beneficial for women, employers, and National Health Insurance Bureau.
A formula similar to four-agents-decoction, Toki-shakuyaku-san, which contains three of the four herbs consisted in four-agents-decoction, also shown similar effectiveness in the treatment of dysmenorrhea among 40 subjects who were equally divided into two groups (11). The dose of the medicine in this double-blind clinical trial was 7.5 g/day, taken three times daily. The trial took a period of 6 menstrual cycles, including 2 cycles each of wash-out, treatment, and follow-up. The subjects who had suffered from primary dysmenorrhea for one or more years, and who had a combination of cold, deficiency, yin, and blood stagnation, were selected at the recruitment according to the diagnostic scoring system derived in Japan. Both pain intensity and the consumed amount of rescue analgesics were decreased over the two treatment cycles and significantly lower in the treatment group compared to the placebo group. These differences persisted during the two follow-up cycles after cessation of the treatment.
The cause of primary dysmenorrhea proposed in conventional Western medicine is ischemia in uterine by increased contractility and decreased blood flow from the excessive production of prostaglandins. In traditional Chinese medicine, menstrual pain is thought due to inhibition of qi and blood stasis. The level of PGF 2 α in both peripherial and menstrual blood of dysmenorrheic women was lowered by 21.6% and 38.7%, respectively, after the treatment with Powder of Angelicae Sinensis and Paioniae (當歸芍藥散); whereas no difference was found between pre-and post-treatment in controls (12). While the mechanism of four-agents-decoction in relieving menstraul pain is not clearly known, four-agentsdecoction has been found with high nutrition values of Fe, Zn, Mn, Co, Cd and folic acid, as well as amino acids, such as aspartic acid and arginine (13-15).
Theory of indivisability between Chinese medicine (中醫) and Chinese medicine prescription or formula (中藥) is the fundermental of traditional Chinese medicine; however, the diagnostic system has been mysterious and is considered inaccurate to Western medicine practitioners. A recent study (16) conducted in the US has found that the degree of consistency between diagnoses made by nine practitioners in US, who had been trained at different places, was high only for kidney yin deficiency in 23 women with menopausal symptoms. The high concordance for kidney yin deficiency could be due to high frequency of the sign that is defined broadly as a major sign in differentiated diagnosis; whereas the concordance for other signs or symptoms, which are more complex by the theory of traditional Chinese medicine, was somewhat low. These findings indicate considerable variations exist among practitioners. For menstrual pain, at least seven differentiated pattern systems have been practiced by traditional Chinese medicine practitioners in treatment of dysmenorrhea (17). The inconsistency and variations are often perceived as a result of practitioners' subjective four examiniations (四診): inspection (望診), listening and smelling (聞診), inquiry (問診), and palpation (切診) in determining patterns from differentiated diagnoses,.
Two versions of diagnosis for menstrual pain (18-19) have been officially instituted and are presently practiced in mainland China; no specific guidelines have been published for people living in Taiwan. The scoring systems used in the previously stated study (11) has changed the descriptive qualitative diagnoses of signs and systems into quantitative measures to make the evaluation towards consistency among practitioners and in turn provides more specific diagnosis. Through careful examinations and discussions among a group of traditional Chinese medicine physicians after analyzed by using epidemiologic methods, the differented patterns of individuals may well be determined by scoring symptoms and signs. This diagnosis system for primary dysmenorrhea can be further developed for its entry into clinical practice in Taiwan.

(1) inclusion criteria
Women aged 18 years and older are currently enrolled in China Technology Institute and Takming College, and meet the following criteria: (a) had regular periods (26-33 days) in the past 6 months (b) experienced at least 4 consecutive painful periods (c) pain started one day before or on the onset of bleeding (d) taken analgesics (2) exclusion criteria (a) began experiencing menstrual pain at menarche (b) started having pain more than 3 years after menarche (c) took oral contraceptive pills (d) had sexual life (e) had endometriosis; pelvic inflammatory disease; inflammatory bowel syndromes (f) had gynecological surgery, including pregnancy The study flow is shown in Appendix 1.

A.
Recruitment of Subjects 1. Survey---identification and selection for primary dysmenorrheic women Administering a questionnaire (Appendix 2) to survey 400 women who have experienced menstrual pain in the past six months at China Technology Institute and Takming College. Approval for conducting the survey has been granted by the IRB of NHRI (Appendix 3). The questionnaire will be sent to 400 women by mail. The women can either send the questionnaire back to NHRI or hand it to the nursing teacher, 郝冬梅 or 謝伶俐. The gift, tote bag, will then be given out by the teacher. The data in the questionnaires will be analyzed and examined by the criteria set for this trial. Among those met the criteria listed below (IV-A-2), a total of 150 women initially eligible for the trial are to be recruited.
An informed consent (Appendix 4) will be sent to these 150 women for preview. Later a meeting will be held for these women and their parents or guardians, in which the purposes of the trial will be explained and the procedures of this trial including guidelines of filling out questionnaires, diary and menstrual record will be gone over, and any questions in the content of informed consent will be answered. A signed informed consent will be collected from those interested to participate at the end of meeting. (The contents of the meeting and handbook will be focused on explanation of the trial's characteristics and significance, which will be planned and given by two young physicians, the Co-investigators.) Motrin for 3 days will be given to the participants, which is the only medication and method to be used in wash-out period. These women will then be confirmed for primary dysmenorrhea with ultrasound by gynecologists at Taipei Municipal Chung Hsiao Hospital and Tri-Service General Hospital.

Criteria for pre-screening subjects (Appendix 5) (1) inclusion criteria
Women aged 18 years and older are currently enrolled in China Technology Institute and Takming College, and meet the following criteria: (d) had regular periods (26-33 days) in the past 6 months (e) experienced at least 4 consecutive painful periods (f) pain started one day before or on the onset of bleeding (d) taken analgesics (2) exclusion criteria (g) began experiencing menstrual pain at menarche (h) started having pain more than 3 years after menarche (i) took oral contraceptive pills (j) had sexual life (k) had endometriosis; pelvic inflammatory disease; inflammatory bowel syndromes (l) had gynecological surgery, including pregnancy

Arrangement of Clinic visit
Clinics close to the two colleges are selected. Fifty CTI students go to Taipei Municipal Chung Hsiao Hospital (CHH) and 100 TMC students go to Tri-Service General Hospital (TSGH).
(1) All women will be traced attentively for menstrual cycles by study nurse 賴美齡, the women will be asked to visit hospitals between the 7 th and 12 th days of cycle.
(2) Clinic visits are arranged on one designated morning or afternoon specifically for the study.
(3) Study nurse 賴美齡 will be attending at both hospitals.
(4) All examiners from both hospitals, including study nurse, gynecologists, traditional Chinese medicine physicians, will have regular meetings led by Dr. JY Liu for setting up standardization of procedures, discussing unclear details, and trial operations at clinics. All the contents will be documented as parts of the protocol. (5) All forms and necessary stationary supplies will be furnished by the study center located in 710, Nan-Kang Campus of NHRI. (6) Dr. TW Liu, a consultant of this trial, will provide his experiences in conducting clinical trials to study physicians. He will provide trainings if necessary.
B. Wash-out-one full cycle (WV1) (c) administers questionnaires (Appendix 8) (d) gives out Motrin, copied menstrual record (Appendix 9), and diary (Appendix 10) (e) takes information on emergency or any medical treatment for which women seek on their own (f) makes copy of charts and stores at the study center (g) checks the completion of procedures and records in the logs (Appendix 7 After the confirmation, >100 subjects are enrolled.

D. Grouped Differentiated diagnosis--Ancillary study (see XII)
Drs. 陳建霖、張恒鴻、游智勝、蔡德豐、林高士 Women who have been confirmed for primary dysmenorrheic sufferers will receive differentiated diagnosis performed on by three of the five TCM physicians on Saturdays at Tri-service General Hospital. Each physician will be arranged to perform the diagnosis in similar frequency.

E.
Baseline-one cycle (BV) (For establishing baseline of pain intensity, some subjects may have to wait for one more cycle of wash-out.) 1.
Gynecology examination Drs. 劉裕森 at CHH and 劉嘉燿 at TSGH (a) performs check-up (b) fills out the checklist of menstrual symptoms (Appendix 6) (c) prescribes Motrin for 3 days for rescuing pain in next menstruation (d) records all oral and written communications with other investigators and subjects (e) supports care if emergency arises

2.
Laboratory examinations-all procedures are performed at the respective hospital and all supplies for the collection and shipping of blood specimens are provided by the hospital.

Clinic's Medical Technologists
(a) draw blood 35 ml (2 red, 2 green, and 1 purple top tubes) and perform the following analysis at respective hospital i. CA-125 to exclude endometriosis ii.
Liver function and kidney function for the baseline of toxicity: GOT, GPT, BUN, creatinine, uric acid iii. Anemic condition: hemoglobin, and hematocrit (b) send 1 red top and 1 green top tubes to Taipei Institute of Pathology for testing toxicity of heavy metals: mercury, arsenic, lead, cadmium, and copper, and the test of iron and total binding protein capacity (TIBC)

TCM examination
Drs. 蔡德豐 at CHH and 王麗香 at TSGH (a) diagnoses for differentiated patterns (b) prescribes study products 4. Study nurse 賴美齡: (a) contacts subjects, making sure the subjects are struck by menstrual pain (b) arranges clinic visits for subjects: registration, lead subjects to the clinic, etc. (c) excludes women having CA-125 > 35 or other abnormal values of biochemical parameters shown in the report (d) administers questionnaires (Appendix 8) (e) gives out Motrin, copied menstrual record (Appendix 9), and diary (Appendix 10) (f) takes information on emergency or other medical treatment which women seek on their own (g) makes copy of charts and stores at the study center (h) performs centrifugation of whole blood and aliquots all leftover blood specimens, and brings back to store at study center (i) checks the completion of procedures and records in the logs (Appendix 7) (j) enters data into database (k) delivers study products to the randomized subjects 5. Nursing teacher, 郝冬梅 at CTI and 謝伶俐 at TMC (a) gives out Motrin on gynecologist's order to subjects need extra (b) traces students when necessary (c) collects information on emergency or other medical treatment that subjects have sought on their own Women will be stratified by hospital into two strata and then randomized within each stratum to either treatment or placebo group using the method of block randomization (see VII B) F. Intervention-3 cycles (IV1, IV2, IV3)

Gynecology examination
Drs. 劉裕森 at CHH and 劉嘉燿 at TSGH (a) performs check-up (b) fills out the checklist of menstrual symptoms (Appendix 6) (c) prescribes Motrin for 3 days for rescuing pain in next menstruation (d) records all oral and written communications with other investigators and subjects (e) supports care if emergency arises

2.
TCM examination Drs. 蔡德豐 at CHH and 王麗香 at TSGH: (a) diagnoses for differentiated patterns (b) evaluates the use of study products (c) prescribes study products for subsequent treatment 3. At the 3 rd treatment cycle, another Laboratory Examination-all procedures are performed at the respective hospital and all supplies for collection and shipping of blood specimens are provided by the hospital.

Clinic's Medical Technologists:
(a) draw blood 35 ml (2 red, 2 green, and 1 purple top tubes) and perform the same analysis as at the baseline at respective hospital i. CA-125 ii.
Liver function and kidney function for the baseline of toxicity: GOT, GPT, BUN, creatinine, uric acid iii.
Anemic condition: hemoglobin and hematocrit (b) send 1 red top and 1 green top tubes to Taipei Institute of Pathology for testing toxicity of heavy metals: mercury, arsenic, lead, cadmium, and copper, and the test of iron and TIBC

Study nurse 賴美齡
(a) contacts subjects (b) arranges clinic visits for subjects: registration, lead subjects to the clinic, etc. (c) administers questionnaires (Appendix 8) (d) gives out Motrin, study products, and copied menstrual record (Appendix 9), and diary (Appendix 10) (e) takes information on emergency or any medical treatment for which women seek on their own (f) makes copy of charts and stores at the study center (g) performs centrifugation of whole blood and aliquots all leftover blood specimens, and brings back to store at study center (h) checks the completion of procedures and records in the logs (Appendix 7) (i) enters data into database

Research assistants (1) Research assistant (TBA)
(a) bags the study products, Motrin, menstrual record, and diary (b) hands out the bags to the study nurse (c) collects and record returned study products, menstrual record, and diary (d) manages the inventory of study products on each subject (e) manages files (f) coordinates for the needs between study nurse and subjects (g) arranges meetings (h) enters data and manages database (2) Research assistant 辛毓真 (a) performs quality assurance of four-agents-decoction (b) manages the stock room of study medicines (c) helps bagging (d) helps data entry and management 6. Nursing teacher, 郝冬梅 at CTI and 謝伶俐 at TMC (a) gives out Motrin on gynecologist's order to subjects need extra (b) traces students when necessary (c) collects information on emergency or other medical treatment that subjects have sought on their own A. Grouped differentiated diagnosis will be performed one more time on Saturdays before follow-up period starts and sonography will be at the third treatment visit.
All the procedures used previously will be followed.

Study nurse 賴美齡:
(a) contacts subjects (b) arranges clinic visits for subjects: registration, lead subjects to the clinic, etc. (c) administers questionnaires (Appendix 8) (d) gives out Motrin, copied menstrual record (Appendix 9), and diary (Appendix 10) (e) takes information on emergency or any medical treatment for which women seek on their own (f) makes copy of charts and stores at the study center (g) checks the completion of procedures and records in the logs (Appendix 7) (h) enters data into database

TCM examinations---Ancillary study
starting from the 2 nd follow-up visit, clinics are set on Saturday mornings.

Nursing teacher, 郝冬梅 at CTI and 謝伶俐 at TMC
(a) gives out Motrin on gynecologist's order to subjects need extra (b) traces students when necessary (c) collects information on emergency or other medical treatment that subjects have sought on their own

(三) 試驗期限與進度
An estimate of minimum 15 months is needed to complete this trial. The trial timetable cannot be fixed for the variations of menstrual cycle of participants. Briefly, the preparation will start in July 2002. The recruitment by using survey method will begin in September, 2002 when both returned students and freshmen start the school. Screening will be in the months of October and November, followed by wash-out and baseline periods in December and January. Treatment period will start right after Winter break for three cycles, which will be in the months of February through May, 2003. Follow-up will be from June through September, 2003.

(四) 所需藥品或醫療器材名稱及數量
Study medications include Motrin (400mg) and study products: four-agentsdecoction and placebo. After a preliminary discussions with three traditional Chinese Medicine practitioners individually in the study and an extensive review of literature (Appendix 13), the plan for the treatment has been set tentatively as having subjects take the study products starting seven days prior to and then until the onset of menstruation for one dose a day (26 capsules) and a booster after the end of menstruation for 5 days for each of the three cycles in the treatment period. Warm water should be used when swallowing the capsules. The final dosage will be decided by the investigators after reviewing the results from the survey.
Four-agents-decoction in capsules are made enough for 250 subjects from one batch of raw materials of the four agents: prepared Radix Rehmanniae praeparata, Radix angelicae Sinensis, Radix Paeoniae Alba, and Rhizoma Ligustici Chuanxiong. Each capsule contains 577 mg of powder. Twenty six capsules are needed to be filled for ~15 grams of the powders that are derived from 9 grams of concentrated decoction and 6 grams of starch as excipient from one dosage of raw materials. The number of capsules may be reduced to half, and the process of concentration and quality control of raw materials are under development with supervision offered by the Council of Traditional Chinese Medicine of DOH.

A. Sample Size Calculation
This is a pragmatically oriented pilot trial (20) incorporated with an epidemiologic study. Pain improvement of the two groups was estimated in this pilot study using the data on analgesics for dysmenorrhea in Western medicine. Based on literature on clinical trials of analgesics, the average of pain improvement assessed by response rate was 70% in the treatment group and 30% in the placebo group. This estimate provides 90% power with 5% type I error, the calculated sample size is 56 subjects. With a 25% dropout rate, at least 100 subjects needed to be enrolled into this trial.

B. Randomization and Blindness
Subjects will be grouped into two strata by hospital, CHH for CTI students and TSGH for TMC students, respectively. Within each stratum, the subjects will then be randomized into treatment group or placebo group (Appendix 14). A permuted block randomization scheme is employed to generate random codes and an approximate equal number of subjects for the two treatment groups will be resulted. A list of treatment assignment linked with case number will be generated and kept by the PI and the study statistician. The codes and treatment assignment will not be released to any subjects, staff, and investigators other than those mentioned until the completion of data analysis.

C. Outcome Measures
From main study: 1.
Diary (1) Pain severity and duration for the day (2) Reactions when taking capsules (3) Amount of analgesics used for rescuing the pain (4) Symptoms other than menstrual pain (5) Other medications 2. Menstrual record (1) Menstrual cycle including the dates and heaviness of menses from the first day of the trial (2) Absence from school 3.
Scores of individuals from the ancillary study 2. Questionnaires for pain improvement 3. Blood biochemical measures

From ancillary studies
Variables listed in the Appendix 16-17 will be studied for the relationships with the variables listed in the main studied.
No measure will be estimated by any investigator. All measures will be stratified by the treatment group and the comparisons will be made by groups as well as by changes within individuals by descriptive statistics, parametric or non-parametric analyses, depending on the data type and distribution.

D. Database
A database designed for easy use and reducing errors to minimum in the process of data entry has been proposed to the senior researchers in the division in hope of having it be constructed by the Information Technologist who was newly hired by this division. The database will be located in the study center-710, Nan Kang campus of NHRI. If the idea of having this kind of database cannot be fulfilled, then Excel spreadsheet will be used instead as the means for data storage.

E. Data Management and Analysis
(1) Method of intent to treat is selected for data analysis.
(2) All data will be stratified by treatment group and examined separately. The data will also be evaluated for hospital effect.
The data on characteristics of subjects are extracted out from the first questionnaire. The data of all variables listed in the questionnaires, menstrual record, diary, and checklist of differentiated diagnosis are coded and entered into database by a RA (TBA). All data files will be collated for analysis. The datasets will be reviewed by all investigators, and the access of data will be accommodated to all investigators after sending in a 1-2 pages of proposal for documentation, which should contain the purpose of the access to data, the rationales and methods of the proposed study, and expected results from the study. Statistical analyses will be performed or supervised by the PI and the study statistician using SAS version 8.2 or other statistical softwares.

F. Efficacy and Safety Evaluation
Both diary and menstruation record are filled out before bedtime by subjects. Primary efficacy endpoint: pain improvement Baseline pain score: the score obtained at baseline, or the average score of previous cycles if no pain strikes before baseline cycle.
Pain duration: pain occurs from a day prior to until the second day after the onset of bleeding Pain intensity: 1. the total VAS pain score from a day prior to until the second day after the onset of bleeding 2. the highest VAS pain score (peak of pain) from a day prior to until the second day after the onset of bleeding 3. the mean VAS scores from a day prior to until the second day after the onset of bleeding The three outcomes will also be evaluated by extended measurement of the pain in the entire menstrual cycle.
If a subject discontinues taking the study products but remains to keep up clinic visits will be included in final analysis. To assess placebo effect, subjects are instructed to answer a question on guess of their study assignment in the questionnaire administered at the last follow-up visit and to list the reasons of the guess. The guess could be based on the appearance, taste, and smell of the study products, side effects, or effectiveness of the treatment (Appendix 8). The group of women who make correct guess will be analyzed separately.
The two treatment groups are compared by evaluating pain duration and pain intensity: visual analog scales (VAS), 4-point pain scale, and the amount of consumed analgesics at each of the three intervention and the three follow-up periods. There are following ways for evaluation of pain improvement or positive response:

Pain duration and intensity of the groups
Continuous data: mean and standard deviation, as well as 95% confidence intervals, of pain duration in hour and visual analog scales in mm. Categorical (ordinal) data: frequencies.
(1) duration in hour is stratified into categorical outcome (2) VAS in mm is stratified into categorical outcome.
(3) 4-point pain scale, which remains as in a scale from 1 to 4

Changes in pain intensity within individuals
Continuous data: means and standard deviation of the difference in VAS in mm between baseline and assessed cycle Dichotomous data: response rate by the order of cycles A response of "yes" is defined by (1) positive difference in VAS in at least 18 mm or (2) by a decrease of one scale in categorized pain scales, and "no" by the difference in opposite direction.
If a subject has menstrual pain, regardless of the intensity, in the consecutive five cycles from wash-out to treatment and no pain experienced in two of the three cycles of follow-up periods, then a positive response by her will be taken into account, the frequency of the positive responses by treatment group will in turn be assessed.

Amount of consumed analgesics
A decrease in use of analgesics is to be considered as an efficacy of fouragents-decoction in the treatment of primary dysmenorrhea. Group comparisons as well as the changes within individuals by treatment groups for the amount of consumed analgesics will be performed.
In addition, odds ratios and the corresponding confidence intervals will be evaluated to compare the response rate between treatment groups. Repeated analysis of variance will be used to analyze the effects of treatment within and between groups over the trial period for the outcomes. Adjustment will be made where it is necessary. GEE method will be explored for further evaluation of the efficacy with the above first three approaches, proposed by the study statistician.

Secondary Efficacy endpoint: Association between response and differentiated diagnosis
There will be several patterns derived from the scoring system specified in the Ancillary study which may or may not be ordinal. For each treatment groups, the association between response and the assigned differentiated pattern will be explored by the Fisher's exact test. Appropriate statistical procedures will be determined after discussions among investigators over the final completed dataset.

Safety
The incidence of adverse events, abnormal laboratory values or treatment related toxicity will be summarized by treatment group. The proportions and the 95% confidence intervals will be given. The Pearson's chi-square test and Mantel-Haenszel method will be used to compare the incidence or ordered qualitative outcomes of the two regimens.

G. Interim Analysis and Post Hoc Analysis
Interim analysis is not considered in this trial. If the result of this trial shows positive response, then a dose-response relationship will be explored using stratification by the length of treatment period. Risk factors for dysmenorrhea will be evaluated using the information in the questionnaires from the survey. The frequencies of the number of capsules taken during the treatment period, the types of differentiated diagnosis and food frequency from the survey are compared with the stratified VAS scores, as well as with 4-point pain scales for the assessment of the adequacy of dosage of pain relief and the association with differentiated diagnosis. The adjusted means of continuous data listed in VII-F will be obtained by adjusting for the amount of consumed analgesics. The Degree of agreement among the five Chinese medicine physicians for differentiated patterns will be evaluated by Kappa statistics. The diagnosis scores will be stratified and evaluated for the relationships with the listed variables in VII-F.

(七) 問卷內容(若計畫涉及問卷調查)
Please refer Appendix 2. This questionnaire has been reviewed and approved by the IRB.

七、臨床上不良反應及處理方法：
Ingesting 26 capsules (may be less) per day does not lead to choking, aspiration, or even death, and no severe adverse event or reaction from the study product is anticipated (5). Women are asked to record any discomfort or sickness in diary (Appendix 10) to track the events between administration of the medicine and the adverse reactions.
In the case of emergency or illness, women are asked to seek medical treatment on their own and notify the PI. The women will provide the information to the study nurse or the nursing teacher for record. Consultation from the study physicians will also be available when requested. The study physicians will then decide whether the illness is related to study products.
Subjects are asked to practice absenteeism and avoid becoming pregnant. In the case of pregnancy, the women are asked to discontinue.

2/13/2007
Please refer Appendix 4 and the one in NHRI format.

十一、出產國及核准上市國最高衛生主管機關許可製售證明影印本:
The certificate will be obtained from the manufacture once the company is identified.

Potential Benefits
The direct benefits to the subjects are the opportunity of obtaining the first hand evidence on the efficacy of four-agents-decoction in capsules, differentiated patterns (or scores), and their relationships.

Potential Risks
The risk for the subjects is minimal, if there is any. If subjects experience breakthrough menstrual pain, then analgesics as rescue medicine are supplemented.